The present invention relates to a ligand binding assay for specific assessment of glycosylated hemoglobin.
Proteins in solution in body fluids are continually subject to glycosylation processes. Glucose reacts with the proteins by non-enzymatic reactions to form glycoproteins, and in many cases the level of glycoprotein formation is proportional to the glucose concentration in the body fluid in question. For proteins not initially synthesized as glycoproteins, the fraction of a protein present in glycosylated form is therefore a function of
the life-time of the protein in the organism and
the glucose concentrations to which the protein has been exposed.
Unlike measurements of glucose concentrations in blood, plasma or urine, which only give information about the glucose concentration at the time of sampling, the amount of a protein present in glycosylated form gives an indication of the organism's control of glucose concentration during longer periods of time.
Erythrocytes (red blood cells) have a mean lifetime of approximately 120 days and contain large amounts of haemoglobin. The fraction of erythrocyte haemoglobin in glycosylated form is thus a good measure of the control of the disease in patients with diabetes mellitus, and is a function of the glucose concentrations in the blood of the patient in the weeks prior to the blood sampling.
In clinical practice numerous different methods have been used to measure the glycosylated haemoglobin fraction in order to quantitatively evaluate the long-term control of blood glucose in patients with diabetes mellitus. The main methods which are in clinical use are:
1. Separation of glycosylated and non glycosylated haemoglobins by means of ion-exchange chromatography. This was the first method proposed and still is the clinical method most commonly used. High costs and time-consuming manufacturing methods together with time-consuming performance of the separations are drawbacks of the method, and the results are influenced by small temperature variations.
2. Use of boronic acid derivatives to specifically isolate the glycosylated haemoglobin fraction. It has long been known that boronic acid moieties bind to carbohydrate moieties having cis-diol residues, (Boeseken J., Advances in carbohydrate chemistry 4, 189-210, 1949. Solms J. and Deuel H., Chimica 11, 311, 1957) and this property can be used as the basis for an affinity chromatographic seperation. Thus, for example, boronic acid residues have been chemically immobilized on solid phases such as agarose, for isolation of glycoproteins, carbohydrates and nucleotides (Hageman, J and Kuehn, G., Anal Biochem. 80: 547, 1977.). Columns of such material have also been used to quantify the glycosylated fraction of haemoglobin (Dean P. D. G. & al, UK patent application GB-A-2024829). Such columns are time-consuming and expensive to make and slow to run: Haemolysate must be passed through the column, the different fractions must be collected and the volumes of the fractions must be corrected, before the haemoglobin content of the different fractions can be measured and the fraction of haemoglobin in glycosylated form can be calculated.
3. Electrophoretic separation. Glycosylation of haemoglobin alters the electric charge of the protein, and this may be used to separate the glycosylated and non-glycosylated fractions electrophoretically, following which the different fractions may be quantitated for example by reflectometry. This method is also time-consuming and expensive.
In addition to the glycosylation of haemoglobin within erythrocytes, glycosylation of proteins in serum takes also place at an elevated rate in patients suffering from diabetes mellitus. However, since the different serum proteins have different half-lives in the body, and most of these half-lives are significantly shorter than that of haemoglobin, measurements of glycosylated serum proteins are only used for short to intermediate term retrospective monitoring of the regulation of the disease. Different colorimetric methods for quantitation of glycosylated serum proteins are widely used as an index of diabetic control (Johnsen, Metcalf & Baker, Clinical Chimica Acta 127 (1982) 87-95). However, the quantitation of glycosylated serum proteins has limited clinical value because of the short lifetime of most serum proteins, which is equally true for the glycosylated form. Moreover, from a technical point of view, quantitation of carbohydrates bound to serum proteins requires the collection and preparation of serum which is time-consuming and cumbersome to perform (vein puncture, coagulation for 2 hours, centrifigation and decantation) compared to determination of blood glucose in diabetic patients, which can be performed by microsampling of capillary blood only.
None of the prior art methods for quantitation of glycosylated hemoglobin has become established as an absolute standard method. For one thing, fractions isolated in the different known methods do not exactly correspond ("Measurement of Glycosylated Hemoglobins using affinity chromatography" Bouriotis et al, Diabetologia 21: 579-580, 1981). In ion exchange methods, the fraction named HbAlc is often called "glycosylated hemoglobin", however this fraction may also contain non-glycosylated hemoglobins, and several glycosylated hemoglobins are eluted in different fractions.
Affinity chromatography using immobilized boronic acid residues isolates haemoglobins glycosylated at different residues, including for example haemoglobin glycosylated at lysine residues, in addition to the more common fraction glycosylated at the amino terminal of the .beta.-chain. However, non-glycosylated haemoglobins can also become unspecifically bound to the solid phase used in this method, and not all glycosylated haemoglobin may be bound if the glycosyl moieties are located within the molecule and are not available for binding to solid phases. The different methods for quantitation of glycosylated hemoglobins which are used therefore have different reference range values.
A further problem is that in several methods high levels of glucose will interfere with the quantitation of the glycosylated fraction.
In addition to the said glycosylated fraction of the haemoglobins where glucose is covalently bound to the haemoglobin, there is another glycosylated fraction where the glucose is more loosely bound. These glycosylated haemoglobins are often referred to as "labile" glycohaemoglobins, since their formation may be reversed by washing the erythrocytes or by incubating the erythrocytes in carbohydrate-free solution, or by exposing the glycosylated haemoglobin to pH 5 (Bisse, Berger & Fluckinger, Diabetes 31: 630-633, 1982). However, the geometrical structure of the boronic ester formed with glycosylated haemoglobin is predominantly obtained with the non-reversibly glycosylated forms, and not with the labile preglycosylated form.
As regards alternative forms of assay, immobilization of antibodies on solid phases is commonly used in immunoassay techniques and in immunopurification of proteins and cells. Antibodies may also be used in homogeneous immunoassays, i.e. where both antibodies and antigens are present in solution, and where the antibody/antigen reaction may be measured directly (e.g. by nephelometric or turbidimetric methods) or indirectly (by means of fluorescence or enzyme activation or inhibition). Numerous attempts have been made to use monoclonal and polyclonal antibodies specific for glycosylated haemoglobin, but with limited success. This is mainly due to the fact that most of the carbohydrate residues of glycosylated haemoglobin are present in chemical forms not readily accessible for binding to monoclonal antibodies. Thus, denaturation of the glycosylated haemoglobin is often necessary to achieve such binding, e.g. by adsorption to polystyrene surfaces (Engbaeck F. & al: Clinical Chemistry 35:93-97, 1989) or by chemical denaturation (Knowles 7 al: U.S. Pat. No. 4,658,022, 1987).
Burnett J. B. & al (Biochemical and Biophysical Research Communication, vol. 96, p. 157-162, 1980) have synthesized a fluorescent boronic acid molecule, N-(5-dimethylamino-1-naphthalene sulfonyl)-3-aminobenzene boronic acid, which was demonstrated to bind to cell membranes for fluorescent microscopy of cells.
Fluorescent and coloured diazo conjugates of boronic acids for quantification of total glycosylated proteins present in a sample were described by Schleicher in German patent application DE 3720736, published Jan. 5, 1989. The Schleicher method relies on one of the following three principles for measurement of total glycoprotein present in a sample:
1. A shift in the absorption maximum of the boronic acid conjugate when bound to glycosylated moities of glycoproteins present, or
2. a polarization change of fluorescence when the fluorescent boronic acid conjugates bind to glycosylated moities of the total glycoproteins present, or
3. measuring the amount of the coloured or fluorescent boronic acid conjugate bound to the total glycoprotein present after removal of excess unbound boronic acid conjugate, for example using activated charcoal.
None of the methods described by Schleicher (ibid) can however be used for the quantitation of a specific glycoprotein in a mixture of other glycoproteins, and especially not for the specific determination of glycosylated haemoglobin in haemolysate samples from patients. In addition, the reagents described by Schleicher have rather low absorption coeffecients and their absorption maxima lie close to those of haemoglobin, making it difficult or impossible to detect glycosylated haemoglobin even when it is present in pure form. Indeed Schleicher makes no mention of the use of his method for the analysis of glycosylated haemoglobin, but proposes instead the analysis of total serum glycosylated proteins and glycosylated albumin extracted from human serum.
Lectins reactive to glycoproteins have also been investigated, although so far no lectins specifically reactive to glycosylated hemoglobin and useful in the quantitation of glycosylated haemoglobins have been identified.
A need therefore exists for an improved method of assaying glycosylated haemoglobin which is specific, rapid, simple to use and readily adapted for use in clinical laboratories.